Enzymology of the Cyclodextrins

Summary

The cyclization reaction is a special type of the 4-α-D-gluco-pyranosyltransfer reactions typical of the cyclodextrin glyco-syltransferases (CGTs). The enzyme from K.pneumoniae M 5 al simultaneously catalyzes cyclization and an acceptor-dependent transfer of linear fragments of 4-α-D-glucopyranosyl chains (disproportionation). Maximum cyclization rates are obtained with chain lengths of glc₁₆-glc₈₀, indicating the dependence upon the helical conformation of the substrate. αCD is formed initially by most of the CGTs. The rates of βCD- and γCD-for-mation are extremely low. In contrast to αCD the higher CDs scarcely participate in reverse reactions and, therefore, accumulate. Only the incubation time determines which CD is obtained as the main cyclic product. By shifting the reaction equilibrium in the cyclization direction the yields of CDs can be enlarged significantly. Using an ultra-/diafiltration reactor, 70% of starch (enzyme-substrate ratio w/w) 1:100,000) can be converted into CDs.

Essentially 6 bacterial strains (4 bacilli) are known for producing extracellular CGTs. Depending upon the strain and the cultural conditions, 5–430mg of enzyme per 1 of culture filtrate were obtained, and purified up to 140fold (overall yields 10–78%). Because of the different assay methods the CGTs of the various bacterial sources cannot be compared at present. It cannot be decided, therefore, which enzyme is the most suited for the production of CDs on a technical scale.

The history of .cyclodextrin glycosyltransferase -research is a very strange one for an enzymologist by several reasons: a) For 70 years only one organism (Bacillus macerans) has been known for producing this type of enzyme. b) Quantitative in­vestigations of the chemistry of the cyclodextrin glycosyl­transferases (1,4-a-D-glucan:[1,4-a-D-glucopyranosyl] transferases (cyclizing) (EC 2.4.1.19), CGTs) have been severely handi­capped by the lack of a specific assay method. c) As indicated by more than 600 publications and patents (1) the chemists have been fascinated by the clathrate-forming properties of the cha­racteristic cyclic transfer products. In spite of the fact that the CGTs are very interesting enzymes, the investigations of both the enzyme chemistry and the mechanism of the reactions have been neglected.

According to D.French, one of the pioneers in CGT-research, “the Schardinger dextrins could be manufactured on a very large scale, if there were a suitable market for them”(2). Up to now, the cyclodextrins (CDs) have been much too expensive for appli­cation on a technical scale. Production of CDs of low costs de­pends upon both an easily available CGT of wellknown characte­ristics and economic methods for manufacturing the cyclic com­pounds.